SYBR Green I-based qPCR reagent: It contains PowerPCR™ Hot Start Taq DNA polymerase, PCR buffer, dNTPs, SYBR Green I fluorescent dyes and Mg2+. It can be applied to the target sequence detection of genomic DNA and cDNA. The SYBR Green I dye binds to double-stranded (ds) DNA without using sequence-specific probes. The chemically modified PowerPCR™ Hot Start Taq DNA polymerase is inactive at room temperature which effectively avoids the nonspecific amplification caused by primer dimers or the nonspecific binding of prime and template. The enzyme should be activated by incubation at 95°C for 10 minutes. The unique PCR buffer components and the hot start enzyme ensure PCR specificity and sensitivity. It has a higher sensitivity and a wider linear range which can exactly quantitate the target gene or the low copy template.

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SYBR Green I-based qPCR reagent: It contains PowerPCR™ Hot Start Taq DNA polymerase, PCR buffer, dNTPs, SYBR Green I fluorescent dyes, Mg2+ and ROX calibration dye. It can be applied to the target sequence detection of genomic DNA and cDNA. The SYBR Green I dye binds to dsDNA without using sequence-specific probes. The chemically modified PowerPCR™ Hot Start Taq DNA polymerase is inactive at room temperature which effectively avoids the non-specific amplification caused by primer dimers or the nonspecific binding of prime and template. The enzyme should be activated by incubation at 95°C for 10 minutes. The unique PCR buffer components and the Hot start enzyme ensure PCR specificity and sensitivity. The ROX is a dye molecule that can be used to normalize the well-to-well fluorescent fluctuations and can be applied to all qPCR instruments using ROX as calibration dye. It has a higher sensitivity and a wider linear range which can exactly quantitate the target gene or low copy template. Suitable Real Time PCR Detection Systems ABI Prism 7500/7500 Fast Strategene MX3000/MX3005P Corbett Rotor Gene 3000 Systems need low ROX signals

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SYBR Green I-based qPCR reagent: It contains PowerPCR™ Hot Start Taq DNA polymerase, PCR buffer, dNTPs, SYBR Green I fluorescent dyes, Mg2+ and ROX calibration dye. It can be applied to the target sequence detection of genomic DNA and cDNA. The SYBR Green I dye binds to dsDNA without using sequence-specific probes. The chemically modified PowerPCR™ Hot Start Taq DNA polymerase is inactive at room temperature which effectively avoids the non-specific amplification caused by primer dimers or the nonspecific binding of prime and template. The enzyme should be activated by incubation at 95°C for 10 minutes. The unique PCR buffer components and the Hot start enzyme ensure PCR specificity and sensitivity. The ROX is a dye molecule that can be used to normalize the well-to-well fluorescent fluctuations and can be applied to all qPCR instruments using ROX as calibration dye. It has a higher sensitivity and a wider linear range which can exactly quantitate the target gene or low copy template. Suitable Real-Time PCR detection systems: ABI Prism7000/7300/7700/7900 ABI Step One/StepOne Plus Eppendorf systems need high ROX signals

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Intended Use The 2x Fast qPCR Mastermix is used for real-time qualitative and quantitative qPCR with SYBR Green dye. ; The Mastermix is a premixed, 2x concentrated solution that has all the components except for gene-specific primers, probes and templates Kit Characterizations The kit is designed for a single gene qPCR with SYBR Green dye. The kit contains Taq-Fast DNA polymerase which can extend more than 300 bases with a short cycling program. The concentrations of the primers and probes are variable depending on specific assays and thermocycling protocols (Table 1). The preferable PCR product size is ≤150bp. The kit has three formulations of No ROX, Low ROX or High ROX concentrations for corresponding instruments(see Table 4). Transportation and storage The kit can be transported at below 4°C for up to 3 days. The kit should be kept stable in the dark at -20°C for ≤24 months with ≤10 times of freeze-thaw cycles. The kit can be stored at 4°C for a week. Setup Reaction and Thermocycling Thaw 2x Mastermix and other reaction components at room temperature, mix each component, centrifuge and then place on ice. Set up reactions (Table 1). Seal tubes with flat, optically transparent caps or seal plate with optically transparent film. Mix and then briefly centrifuge the tubes or plate. Program PCR instrument with indicated thermo-cycling protocol. Load PCR tubes or plate and start to run. Perform data analysis according to the PCR instrument instructions. Table 1. Setting up a 20μl or 10μl Reaction Component Volume per 20μl Volume per 10μl Final Concentration 2x Mastermix 10μL 5μL 1X Primersa Variable Variable Each 150-900nM DNA Templateb Variable Variable ≤30ng genomic DNA/10μL H2O To 20μL To 10μL Note: Each primer’s Tm should be designed ≥60°C, preferably between 62°C to 65°C, using primer3 software for high efficiency and specificity. DNA templates should be extracted by a qualified silica-based kit and eluted with low EDTA TE buffer (10mM Tris-HCl, 0.1mM EDTA, pH 8.0-8.3). Table 2. Standard Thermocycling Protocol Stage Temperature Period Number of Cycles I 95°C 2 min 1 II 95°C 12 sec 35-40 II 60°C, signal acquisition 60 sec 35-40 III 60°C to 95°C  Various 1 Note: The primer concentration used is typically 0.2uM Table 2. Standard Thermocycling Protocol Stage Temperature Period Number of Cycles I 95°C 1 min 1 II 95°C 5 sec 35-40 II 60°C, signal acquisition 30 sec 35-40 III 60°C to 95°C  Various 1 Note:  The product size for the fast thermocycling protocol is preferred to be less than 90bp. The primer concentration used is typically between 0.4uM and 0.9uM. Table 4. Compatible Instruments qPCR Instrument ROX required by instrument Passive dye setup Bio-Rad® iQ™5, CFX96, CFX384, Opticon Roche Lightcycler®Qiagen Rotor-Gene™Eppendorf Mastercycler®Cepheid® SmartCycler® Not recommended Not necessary Applied Biosystems® 7500, 7500 Fast, QuantStudio™, ViiA7™, Agilent Mx™ Low ROX(50nM final concentration) Turn on ROX passive reference dye  Applied Biosystems® 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne™, StepOnePlus™ High ROX(500nM final concentration) Turn on ROX passive reference dye    Quality Control  Not detectable DNase and RNase contaminations.  Precautions  If you order a “No ROX” master mix but you have an Applied Biosystems or ThermoFisher instrument, please turn off ROX passive reference dye button when setup assays.

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Intended Use The 2x Fast qPCR Mastermix is used for real-time qualitative and quantitative qPCR with SYBR Green dye. ; The Mastermix is a premixed, 2x concentrated solution that has all the components except for gene-specific primers, probes and templates Kit Characterizations The kit is designed for a single gene qPCR with SYBR Green dye. The kit contains Taq-Fast DNA polymerase which can extend more than 300 bases with a short cycling program. The concentrations of the primers and probes are variable depending on specific assays and thermocycling protocols (Table 1). The preferable PCR product size is ≤150bp. The kit has three formulations of No ROX, Low ROX or High ROX concentrations for corresponding instruments(see Table 4). Transportation and storage The kit can be transported at below 4°C for up to 3 days. The kit should be kept stable in the dark at -20°C for ≤24 months with ≤10 times of freeze-thaw cycles. The kit can be stored at 4°C for a week. Setup Reaction and Thermocycling Thaw 2x Mastermix and other reaction components at room temperature, mix each component, centrifuge and then place on ice. Set up reactions (Table 1). Seal tubes with flat, optically transparent caps or seal plate with optically transparent film. Mix and then briefly centrifuge the tubes or plate. Program PCR instrument with indicated thermo-cycling protocol. Load PCR tubes or plate and start to run. Perform data analysis according to the PCR instrument instructions. Table 1. Setting up a 20μl or 10μl Reaction Component Volume per 20μl Volume per 10μl Final Concentration 2x Mastermix 10μL 5μL 1X Primersa Variable Variable Each 150-900nM DNA Templateb Variable Variable ≤30ng genomic DNA/10μL H2O To 20μL To 10μL Note: Each primer’s Tm should be designed ≥60°C, preferably between 62°C to 65°C, using primer3 software for high efficiency and specificity. DNA templates should be extracted by a qualified silica-based kit and eluted with low EDTA TE buffer (10mM Tris-HCl, 0.1mM EDTA, pH 8.0-8.3). Table 2. Standard Thermocycling Protocol Stage Temperature Period Number of Cycles I 95°C 2 min 1 II 95°C 12 sec 35-40 II 60°C, signal acquisition 60 sec 35-40 III 60°C to 95°C  Various 1 Note: The primer concentration used is typically 0.2uM Table 3. Fast Thermocycling Protocol  Stage Temperature Period Number of Cycles I 95°C 1 min 1 II 95°C 5 sec 35-40 II 60°C, signal acquisition 30 sec 35-40 III 60°C to 95°C  Various 1 Note:  The product size for the fast thermocycling protocol is preferred to be less than 90bp. The primer concentration used is typically between 0.4uM and 0.9uM. Table 4. Compatible Instruments qPCR Instrument ROX required by instrument Passive dye setup Bio-Rad® iQ™5, CFX96, CFX384, Opticon Roche Lightcycler®Qiagen Rotor-Gene™Eppendorf Mastercycler®Cepheid® SmartCycler® Not recommended Not necessary Applied Biosystems® 7500, 7500 Fast, QuantStudio™, ViiA7™, Agilent Mx™ Low ROX(50nM final concentration) Turn on ROX passive reference dye  Applied Biosystems® 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne™, StepOnePlus™ High ROX(500nM final concentration) Turn on ROX passive reference dye    Quality Control  Not detectable DNase and RNase contaminations.  Precautions  If you order a “No ROX” master mix but you have an Applied Biosystems or ThermoFisher instrument, please turn off ROX passive reference dye button when setup assays.

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Intended Use The 2x Fast qPCR Mastermix is used for real-time qualitative and quantitative qPCR with SYBR Green dye. ; The Mastermix is a premixed, 2x concentrated solution that has all the components except for gene-specific primers, probes and templates Kit Characterizations The kit is designed for a single gene qPCR with SYBR Green dye. The kit contains Taq-Fast DNA polymerase which can extend more than 300 bases with a short cycling program. The concentrations of the primers and probes are variable depending on specific assays and thermocycling protocols (Table 1). The preferable PCR product size is ≤150bp. The kit has three formulations of No ROX, Low ROX or High ROX concentrations for corresponding instruments(see Table 4). Transportation and storage The kit can be transported at below 4°C for up to 3 days. The kit should be kept stable in the dark at -20°C for ≤24 months with ≤10 times of freeze-thaw cycles. The kit can be stored at 4°C for a week. Setup Reaction and Thermocycling Thaw 2x Mastermix and other reaction components at room temperature, mix each component, centrifuge and then place on ice. Set up reactions (Table 1). Seal tubes with flat, optically transparent caps or seal plate with optically transparent film. Mix and then briefly centrifuge the tubes or plate. Program PCR instrument with indicated thermo-cycling protocol. Load PCR tubes or plate and start to run. Perform data analysis according to the PCR instrument instructions. Table 1. Setting up a 20μl or 10μl Reaction Component Volume per 20μl Volume per 10μl Final Concentration 2x Mastermix 10μL 5μL 1X Primersa Variable Variable Each 150-900nM DNA Templateb Variable Variable ≤30ng genomic DNA/10μL H2O To 20μL To 10μL Note: Each primer’s Tm should be designed ≥60°C, preferably between 62°C to 65°C, using primer3 software for high efficiency and specificity. DNA templates should be extracted by a qualified silica-based kit and eluted with low EDTA TE buffer (10mM Tris-HCl, 0.1mM EDTA, pH 8.0-8.3). Table 2. Standard Thermocycling Protocol Stage Temperature Period Number of Cycles I 95°C 2 min 1 II 95°C 12 sec 35-40 II 60°C, signal acquisition 60 sec 35-40 III 60°C to 95°C  Various 1 Note: The primer concentration used is typically 0.2uM Table 3. Fast Thermocycling Protocol  Stage Temperature Period Number of Cycles I 95°C 1 min 1 II 95°C 5 sec 35-40 II 60°C, signal acquisition 30 sec 35-40 III 60°C to 95°C  Various 1 Note:  The product size for the fast thermocycling protocol is preferred to be less than 90bp. The primer concentration used is typically between 0.4uM and 0.9uM. Table 4. Compatible Instruments qPCR Instrument ROX required by instrument Passive dye setup Bio-Rad® iQ™5, CFX96, CFX384, Opticon Roche Lightcycler®Qiagen Rotor-Gene™Eppendorf Mastercycler®Cepheid® SmartCycler® Not recommended Not necessary Applied Biosystems® 7500, 7500 Fast, QuantStudio™, ViiA7™, Agilent Mx™ Low ROX(50nM final concentration) Turn on ROX passive reference dye  Applied Biosystems® 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne™, StepOnePlus™ High ROX(500nM final concentration) Turn on ROX passive reference dye    Quality Control  Not detectable DNase and RNase contaminations.  Precautions  If you order a “No ROX” master mix but you have an Applied Biosystems or ThermoFisher instrument, please turn off ROX passive reference dye button when setup assays.

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